Sequence-selective assembly of tweezer-molecules on linear templates enables frameshift-reading of sequence information

Monomer-sequence information in synthetic copolyimides can be recognised by tweezer-type molecules binding to adjacent triplet-sequences on the polymer chains. In the present paper different tweezer-molecules are found to have different sequence-selectivities, as demonstrated in solution by 1H NMR spectroscopy and in the solid state by single crystal X-ray analyses of tweezer-complexes with linear and macrocyclic oligo-imides. This work provides clear-cut confirmation of polyimide chain-folding and adjacent-tweezer-binding. It also reveals a new and entirely unexpected mechanism for sequence-recognition which, by analogy with a related process in biomolecular information processing, may be termed "frameshift-reading". The ability of one particular tweezer-molecule to detect, with exceptionally high sensitivity, long-range sequence-information in chain-folding aromatic copolyimides, is readily explained by this novel process.

Aberrant mRNA Transcripts and the Nonsense-Mediated Decay Proteins UPF2 and UPF3 Are Enriched in the Arabidopsis Nucleolus

The eukaryotic nucleolus is multifunctional and involved in the metabolism and assembly of many different RNAs and ribonucleoprotein particles as well as in cellular functions, such as cell division and transcriptional silencing in plants. We previously showed that Arabidopsis thaliana exon junction complex proteins associate with the nucleolus, suggesting a role for the nucleolus in mRNA production. Here, we report that the plant nucleolus contains mRNAs, including fully spliced, aberrantly spliced, and single exon gene transcripts. Aberrant mRNAs are much more abundant in nucleolar fractions, while fully spliced products are more abundant in nucleoplasmic fractions. The majority of the aberrant transcripts contain premature termination codons and have characteristics of nonsense-mediated decay (NMD) substrates. A direct link between NMD and the nucleolus is shown by increased levels of the same aberrant transcripts in both the nucleolus and in Up-frameshift (upf) mutants impaired in NMD. In addition, the NMD factors UPF3 and UPF2 localize to the nucleolus, suggesting that the Arabidopsis nucleolus is therefore involved in identifying aberrant mRNAs and NMD.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E-coli O157 : H7

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

The variant rpoS allele of S-enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::str(r) null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::str(r). Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.